Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a protein kinase A (PKA)-activated, ATP-gated chloride channel. Defective function of this channel in the apical membrane of epithelial cells is responsible for the debilitating symptoms in patients with cystic fibrosis. Although PKA-dependent phosphorylation of the regulatory (R) domain of CFTR is critical for CFTR function, the molecular mechanism of how phosphorylation of the R domain activates CFTR remains unclear. How many serine residues need to be phosphorylated to activate CFTR? Which residues are essential? Are those phosphorylation sites functionally degenerate or distinct? Biophysical studies of CFTR modulation by pharmacological reagents have led to the conclusion that membrane bilayer properties play a critical role in CFTR function. Pilot studies show that cholesterol, a key lipid component in cell membranes, has a major impact on CFTR function and its response to pharmacological reagents. How does cholesterol affect CFTR? Does it bind to the CFTR protein? Is the effect of cholesterol on CFTR gating secondary to an alteration of membrane fluidity? Is the choleserol-rich microdomain of the cell membrane involved? A multi-disciplinary team with biochemist, biophysicist, bioengineer and molecular biologist has been assembled to tackle these important questions. A variety of techniques will be used including site-directed mutagenesis, cell-attached, excised inside-out and whole-cell configurations of the patch-clamp technique, rapid photorelease of caged cAMP and membrane fluidity measurements with novel molecular rotors. The proposal is aimed to 1) study the molecular basis for phosphorylation-dependent regulation of CFTR function, and 2) investigate the biophysical and biochemical mechanisms for CFTR modulation by cholesterol. A clear picture of how CFTR is regulated by phosphorylation machinery and lipid environment will emerge from our studies. The information obtained will not only facilitate a fundamental understanding of how CFTR functions, but also aid in the development of novel therapeutics for patients with cystic fibrosis.